Thrombin, the terminal serine proteinase of the blood coagulation cascade, plays a pivotal role in thrombus formation as well as its regulation. These multiple and sometimes opposing roles of thrombin in coagulation are influenced by two exosites (ABE1 and ABE2) on opposite faces of the enzyme and cofactors or ligands that bind to these or other sites to allosterically regulate the proteinase. Major gaps and inconsistencies remain in the mechanistic understanding of how thrombin allostery is achieved and translates into regulated function. We broach the problem with the unexpected finding that thrombin can readily and reversibly interconvert along a continuum of zymogen-like and proteinase-like states depending on the complement of ligands bound to the enzyme. We propose that these interconversions lie at the heart of thrombin allostery. Our studies center on the finding that fragment 1.2 (F12), the authentic protein ligand for ABE2, thermodynamically favors zymogen- like forms. We will now employ titration calorimetry to establish the thermodynamic basis by which thrombomodulin (TM), which binds to ABE1, selectively stabilizes and favors proteinase-like forms to oppose the effects of F12. We also find that meizothrombin (mIIa), produced as an intermediate during thrombin formation, is particularly zymogen-like because of covalent linkage between F12 and the proteinase domain. We will employ a stopped-flow kinetic approach to examine the distribution of mIIa between zymogen-like and proteinase-like forms. Although the F2 region within F12 is expected to contact ABE2, we now propose a novel function for the Ca2+ stabilized structure of the putatively distant F1 in enforcing the ability of F12 to favor zymogen-like forms. Based on these mechanistic studies, we will study the regulation of thrombin function by opposing effects of ABE1 binding by different ligands and F12 binding to ABE2, with an eye to explaining the apparent differences in specificity ascribed to mIIa and various anticoagulant thrombins. We hypothesize that their altered specificity lies in their significantly zymogen-like character that can be variably rescued by the binding of ligands and substrates. These concepts also provide the appropriate framework for the development of novel aptamer probes that can modulate the distribution of thrombin between zymogen-like and proteinase- like states and thereby regulate its specificity with therapeutic potential. Finally, based on our recent successful entry into the structural biology arena, we propose the long term goal of solving a series of novel structures to establish the structural basis for the zymogenizing function of F12. Our strategies bring fresh and unifying concepts to the important problem of thrombin allostery. We anticipate our findings to shed new light on the mechanisms at play in regulating thrombin function in normal hemostasis and in disease states. Our findings have the potential to reveal new strategies for therapeutic targeting of this enzyme in thrombotic and vascular disease. PUBLIC HEALTH RELEVANCE: Thrombin is the pivotal enzyme produced in blood that both accelerates and slows down the formation of the blood clot. Our research brings new concepts and methodologies to bear on solving how thrombin participates in multiple reactions with opposing consequences. Our findings will reveal novel strategies for selectively interfering with the different functions of thrombin for the treatment of life-threatening blood clots in a number of human diseases.